WO2000010588A2 - Inhibition de la production de mucus dans les voies respiratoires par l'administration d'antagonistes d'egf-r - Google Patents

Inhibition de la production de mucus dans les voies respiratoires par l'administration d'antagonistes d'egf-r Download PDF

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WO2000010588A2
WO2000010588A2 PCT/US1999/018696 US9918696W WO0010588A2 WO 2000010588 A2 WO2000010588 A2 WO 2000010588A2 US 9918696 W US9918696 W US 9918696W WO 0010588 A2 WO0010588 A2 WO 0010588A2
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egf
cells
antagonist
goblet
antibody
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PCT/US1999/018696
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WO2000010588A9 (fr
WO2000010588A3 (fr
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Jay A. Nadel
Kiyoshi Takeyama
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The Regents Of The University Of California
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Priority to EP99943729A priority Critical patent/EP1119349B1/fr
Priority to PL351765A priority patent/PL200940B1/pl
Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Priority to SK216-2001A priority patent/SK287805B6/sk
Priority to AU56766/99A priority patent/AU760766B2/en
Priority to DK99943729T priority patent/DK1119349T3/da
Priority to SI9931016T priority patent/SI1119349T1/sl
Priority to EEP200100097A priority patent/EE200100097A/xx
Priority to UA2001021134A priority patent/UA73722C2/uk
Priority to BR9912672-9A priority patent/BR9912672A/pt
Priority to HU0103894A priority patent/HUP0103894A3/hu
Priority to JP2000565908A priority patent/JP2002539072A/ja
Priority to DE69939022T priority patent/DE69939022D1/de
Priority to IL14085599A priority patent/IL140855A0/xx
Priority to CA2337422A priority patent/CA2337422C/fr
Priority to NZ509271A priority patent/NZ509271A/en
Priority to EA200100136A priority patent/EA004316B1/ru
Publication of WO2000010588A2 publication Critical patent/WO2000010588A2/fr
Publication of WO2000010588A9 publication Critical patent/WO2000010588A9/fr
Priority to IL140855A priority patent/IL140855A/en
Priority to BG105158A priority patent/BG105158A/bg
Priority to NO20010749A priority patent/NO327000B1/no
Priority to HR20010119A priority patent/HRP20010119B1/xx
Publication of WO2000010588A3 publication Critical patent/WO2000010588A3/fr
Priority to HK01106832A priority patent/HK1036219A1/xx

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/08—Bronchodilators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/10—Expectorants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/12—Mucolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators

Definitions

  • This invention relates generally to the field of pulmonary treatment More particularly, the invention relates to inhibiting hypersecretion of mucus in lungs and airways by the administration of an EGF-R antagonist In addition, this invention also relates to methods for the development or assessment of candidate agents capable of inhibiting hypersecretion of mucus in the lungs
  • the mucociliary system serves as the primary defense mechanism to move inhaled particles or infectious agents out of the airways in the lungs
  • substances present in airway fluids serve to limit the toxicity of the particles and to inactivate infective agents
  • the physical mechanism of coughing serves to expel the mucus from the airway passages (see e g , "Foundations of Respiratory Care,” Pierson and Kacmare , eds (1992) Churchill Livingstone Inc New York, New York, “Harrison's Principles of Internal Medicine", Fauci et al , eds (1997) 14th Edition, McGraw Hill, New York, New York)
  • the mucociliary system consists of ciliated epithelial cells, epithelial goblet cells, and serous and mucous cells located in submucosal glands
  • the cilia are surrounded by an aqueous layer (penciliary fluid) secreted into the lumen of the airway passage by the active transport of chloride and the passive movement of water across the epithelium
  • the cilia make contact with the mucus floating on this aqueous layer, and via a unidirectional propelling motion provide for movement of mucus toward the glottis (see Pierson and Kacmarek, supra and Fauci, ef al , supra)
  • Mucus is produced by the epithelial goblet cells and submucosal gland cells and is secreted into the lumen of the airway after degranulation
  • ulcerative colitis Hypersecretion of mucus is the major symptom in patients with chronic obstructive pulmonary disease (COPD) and defines the condition (/ e chronic cough and sputum production) This condition alone affects 14 million Americans and can cause progressive disability and death It has been estimated that asthma affects at least 4% of the U S population and accounts for at least 2000 deaths annually (Pierson and Kucmarek, supra) During an acute asthmatic event, the bronchial walls swell, mucus volume increases and bronchial smooth muscle contracts, resulting in airway narrowing. As a result of hypersecretion in acute asthma, extensive mucus plugging can be a major cause of morbidity and mortality
  • cystic fibrosis is an autosomal recessive disease that causes the airway mucosal cell to become unresponsive to cychc-AMP-dependent protein kinase activation of the membrane chloride ion channels (Pierson and Kacmarek, supra and Fauci, ef al , supra)
  • the subsequent electrolyte imbalance reduces the level of hydration of the airway mucus, thus resulting in highly viscous mucus in the lungs of an individual afflicted with cystic fibrosis
  • Hypersecretion obstructs the air passages of individuals with cystic fibrosis, further compromising lung function
  • Other disease involving hypersecretion include chronic obstructive lung disorder (COPD)
  • Mechanical intubation is often necessary in order to provide assisted ventilation to patients with various pulmonary diseases
  • a tube is introduced via the oropharanx and placed in the trachea
  • a balloon is inflated around the tube in the lower trachea, which may abrade the epithelium and cause goblet cell metaplasia Wounding of epithelium leads to repair processes, which can result in abundant mucus secretion
  • Such prolonged tracheal intubation in patients can lead to deleterious effects due to hypersecretion
  • bronchodilators e g , methylxanthines, sympathomimetics with strong ⁇ 2 adrenergic stimulating properties, anticholmergics
  • systemic or inhaled corticosteroids primarily in asthma, liquefaction of the mucus by oral administration of expectorants, e g guaifenesin, and aerosol delivery of "mucolytic" agents, e g water, hypertonic saline solution
  • a newer therapy for cystic fibrosis is the administration of DNAse to target the DNA- ⁇ ch mucus or sputum (Shak, ef al (1990) Proc Natl Acad (USA) 87 9188-9192, Hubbard, R C et al (1991) N Engl J Med 326 812)
  • a primary object of the invention is to provide a method of treating diseases involving hypersecretion of mucus in lungs
  • Another object of the invention is to provide formulations useful in the treatment of diseases that result in hypersecretion of mucus
  • Yet another object of the invention is to provide an in vitro assay for the screening of candidate agents that inhibit hypersecretion of mucus, where the method involves the steps of (i) contacting an in vitro model of goblet cell proliferation with EGF or the functional equivalent thereof, (n) subsequently contacting the in vitro model with a candidate agent, and (m) assessing goblet cell proliferation, wherein inhibition of goblet cell proliferation is indicative of the candidate agent's therapeutic potential
  • Another object of the invention is to provide an in vivo assay for the screening of candidate agents that inhibit hypersecretion of mucus, where the method involves (i) creating an animal model of hypersecretory pulmonary disease by inducing EGF-R, e g with tumor necrosis factor-alpha (TNF- ⁇ ), (n) stimulating the induced EGF-R with its ligand, e g transforming growth factor alpha (TGF- ) or EGF, to produce mu n producing goblet cells, (in) treating with a candidate agent, and (iv) assessing goblet cell proliferation or mucus secretion, wherein an inhibition of goblet cell proliferation or mucus secretion is indicative of the candidate agent's therapeutic potential
  • a further object of the invention is to provide in vitro and in vivo assays for the screening of EGF-R antagonists that inhibit hypersecretion of mucus
  • An advantage of the invention is that it provides a means for preventing excessive formation of mucus in pulmonary airways
  • a feature of the invention is that a range of different types of antagonists can be used to block the effects of EGF and/or TGF- ⁇ and their interaction with EGF-R
  • An aspect of the invention is formulations of EGF antagonists for reducing formation of mucus secretion in the airways of a mammalian patient, preferably a human patient.
  • Another object of the invention is a method of pulmonary delivery of EGF antagonists for reducing mucus secretions in the airways of a mammalian patient, preferably a human patient.
  • Another object of the invention is to provide a method for treating a range of different diseases which have as a symptom the excess formulation of mucus secretions in the airways. These diseases include, without limitation, chronic bronchitis, acute asthma, cystic fibrosis, bronchiectasis, chronic obstructive lung disease, hypersecretion resulting from epithelial damage such as allergic stimuli or mechanical abrasions, and nasal hypersecretion.
  • Figure 1A is a western blot of EGF-R in NCI-H292 and in A431 cells.
  • Figure 1B immunocytochemical analysis with anti-EGF-R antibody in cultures of NCI-H292 cells.
  • Figure 1C Northern analysis of EGF-R in NCI-H292 cells.
  • Figure 2 Alcian blue/PAS staining of NCI-H292 cells for identification of mucin glycoproteins.
  • FIG 4A and 4B Immunohistochemical analysis of EGF-R with an anti-EGF-R antibody in pathogen-free rats.
  • Figure 4A TNF ⁇ -treated rats.
  • Figure 4B ovalbumin-sensitized rats.
  • Figure 5 is a graph depicting the effect of EGF-R tyrosine kinase inhibitor (BIBX1522) on production of goblet cells (expressed as % of stained area of airway epithelium occupied by Alcian blue/PAS-positive stained cells).
  • compositions and methods are provided for the treatment of airway mucus hypersecretion by administering therapeutic amounts of EGF antagonists, preferably kinase inhibitors.
  • EGF antagonists preferably kinase inhibitors.
  • the antagonists may be in the form of small molecules, antibodies, or portions of antibodies that bind to either EGF or its receptor.
  • airway hypersecretory diseases e.g. chronic bronchitis, bronchiectasis, cystic fibrosis, acute asthma, COPD, etc.
  • mucin synthesis in airways is increased and mucus hypersecretion occurs.
  • the secreted mucus results in airway obstruction, an effect that causes death in these diseases.
  • an evolutionary sequence of goblet cell production may be based on the expression of EGF-R Stimulation with TNF ⁇ induces intense EGF-R staining of non-granulated secretory cells, their subsequent activation by EGF-R gands causes progressive staining for mucous glycoconjugates in the cytoplasm, and the cells become "pre-goblet” and then "goblet” cells
  • EGF-R activation promotes selective cell differentiation, but not proliferation
  • Goblet cells are apparently derived from non-granulated secretory cells that express EGF-R and are stimulated by EGF-R gands to produce mucins
  • the EGF-R may be stimulated by other signaling mediators
  • the EGF-R may be stimulated by other signaling mediators
  • Proinflammatory cytokme-activated neutrophils and cigarette smoke are shown to cause mucin synthesis in human bronchial epithelial cells via ligand-independent activation of EGF-R, implicating recruited neutrophils and cigarette smoke as regulators of epithelial cell differentiation that result in abnormal induction of mucin-producing cells in airways
  • Neutrophils activated by a variety of stimuli, including IL-8, N-formyl-methionyl-leucyl- phenylalanine, TNF- ⁇ , cigarette smoke or H 2 0 2 upregulate mucin expression in epithelial cells, which synthesis is inhibited by EGF-R inhibitors
  • Neutrophils are also capable of producing the EGF-R gands, EGF and TGF ⁇
  • epithelial cells including IL-8, N-formyl-me
  • Epithelial damage is a common finding in studies of patients even with mild asthma, and the damage is increasingly related to worsening of clinical symptoms
  • Epithelial damage produced by the allergic response induces EGF-R activation, which results in abnormal goblet cell production
  • EGF-R is implicated in epithelial damage, for example the "airway remodeling" that occurs in asthma, repair and wound closure
  • Mechanical epithelial damage and epithelial injury in asthma may involve a similar EGF-R cascade, resulting in abnormal growth of epithelial secretory cells
  • Hypersecretion is also an important manifestation of inflammatory diseases of the nose
  • nasal goblet cells are "challenged” by inducing goblet cell degranulation utilizing a neutrophil-dependent mechanism, expression of EGF-R and mucins are strongly upregulated These events were associated with regranulation of the goblet cells
  • inflammation such as stimulation of neutrophil infiltration, causes goblet cell degranulation and mucin secretion, up-regulation and activation of EGF-R re-supphes the airway epithelium with mucins
  • EGF epidermal growth factor
  • epidermal growth factor or "EGF” is meant a protein or portion thereof having biological activity characterized by mitogenic activity on epithelial cells (e g , Cohen (1986) Biosciences Reports 6(12) 1017, Aaronson, S A , “Growth Factors and Cancer,” Science (1991 ) 254 1146-1153)
  • epithelial cells e g , Cohen (1986) Biosciences Reports 6(12) 1017, Aaronson, S A , “Growth Factors and Cancer,” Science (1991 ) 254 1146-1153
  • human epidermal growth factor for example as described by Urdea ef al (1983) Proc Nat Acad Sci 80 7461-7465
  • proteins of portions thereof which are the functional equivalent of EGF in terms of the biological response elicited by EGF
  • EGF-R epidermal growth factor receptor
  • a protein a portion thereof capable of binding EGF protein or a portion thereof Exemplary is the human epidermal growth factor receptor (see Ullrich ef al (1984) Nature 309418-425, Genbank accession number NM_005228)
  • the binding of the EGF hgand activates the EGF-R (e g resulting in activation of mtracellular mitogenic signaling, autophosphorylation of EGF-R)
  • EGF-R epidermal growth factor receptor
  • hgands include, but are not limited to, TGF- ⁇ , betacellulin, amphireguhn, hepann-binding EGF (HB-EGF) and neureguhn (also known as hergu n) (Strawn and Shawver (1998) Exp -Opm Invest Drugs 7(4)553-573,
  • EGF-R antagonist any agent capable of directly or indirectly inhibiting the effect of EGF-R, particularly the effect of EGF-R on goblet cell proliferation or hypersecretion of mucus by goblet cells
  • EGF-R can be activated through hgand-dependent and hgand-independent mechanisms, resulting in either autophosphorylation or trans-phosphorylation, respectively
  • EGF-R antagonists of interest may inhibit either or both of these mechanisms
  • binding of TNF- ⁇ to the EGF-R results in a ligand-dependent phosphorylation, which may be blocked by an antibody that binds EGF-R, thereby preventing the interaction of EGF with a hgand that would activate the EGF receptor
  • Examples of such antibodies are described by Goldstein et al (1995) Clin Cancer Res 1 1311-1318, Lo ⁇ mer ef a/ (1995) Clin Cancer Res 1 859-864, Schmidt and Wels (1996) Br J Cancer 74 853-862 Small molecule tyrosine kinas
  • An EGF-R antagonist may be an antibody that binds to a factor that stimulates EGF production or EGF-R production, thereby inhibiting promotion of goblet cell proliferation by EGF (/ e an inhibitor of the phosphorylation cascade that phosphorylates EGF-R)
  • EGF an inhibitor of the phosphorylation cascade that phosphorylates EGF-R
  • a fusion protein of TGFa- Pseudomonas exotoxin 40 is described by Arteaga ef al (1995) Cancer Res 54 4703-4709
  • the EGF-R antagonist is an inhibitor of the tyrosine kinase activity of EGF-R, particularly small molecule inhibitors having selective action on EGF-R as compared to other tyrosine kinases - preferred small molecules block the natural EGF receptor in a mammal, preferably a human and have a molecular weight of less than 1 kD
  • Inhibitors of EGF and EGF-R include, but are not limited to, tyrosine kinase inhibitors such as quinazohnes, such as PD 153035, 4-(3-chloroan ⁇ l ⁇ no) quinazoline, or CP-358,774, pyridopynmidmes, py ⁇ midopy ⁇ midines, pyrrolopy ⁇ midines, such as CGP 59326, CGP 60261 and CGP 62706, and pyrazolopy ⁇ midines (Shawn and Shawver, supra ), 4-(phenylam ⁇ no)-7H-pyrrolo[2,3-d] py ⁇ midines (Traxler ef al , (1996) J Med Chem 39 2285-2292), curcumm (diferuloyl methane) (Laxmin arayana, et al, (1995), Carcinogen 16 1741-1745), 4,5-b ⁇ s (4-fluoroan ⁇ hno)
  • treatment means obtaining a desired pharmacologic and/or physiologic effect
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease
  • Treatment covers any treatment of a disease in a mammal, particularly a human, and includes
  • the invention is directed toward treating patients with pulmonary or airway disease and is particularly directed toward treating patients' hypersecretion of mucus, / e preventing, inhibiting or relieving hypersecretion of mucus
  • the invention is directed toward decreasing mucus or sputum in the airways, inhibiting infection by pathological organisms, alleviating cough, and preventing hypoxia due to airway plugging
  • treatment is intended to mean providing a therapeutically detectable and beneficial effect on a patient suffering from a pulmonary disease involving hypersecretion of mucus
  • treatment shall mean preventing, alleviating, and/or inhibiting hypersecretion of mucus with a compound selected from the group consisting of EGF and/or EGF-R antagonists such as antibodies, protein tyrosine kinase inhibitors and antisense molecules and the like
  • An alternative treatment may comprise prevention of EGF-R expression in airway, thereby blocking the pathway at an earlier stage
  • reagents that block binding of TNF ⁇ to its receptor may prevent upregulation of EGF-R
  • Treatment includes preventing or inhibiting infections by pathological agents caused by and/or related to hypersecretion of mucus
  • antibody an immunoglobu n protein that is capable of binding an antigen
  • Antibody as used herein is meant to include antibody fragments, e g F(ab')2, Fab', Fab, capable of binding the antigen or antigenic fragment of interest
  • the binding of the antibody to the antigen inhibits the activity of EGF or EGF-R
  • humanized antibody is used herein to describe complete antibody molecules, / e composed of two complete light chains and two complete heavy chains, as well as antibodies consisting only of antibody fragments, e g Fab, Fab' , F (ab') 2, and Fv, wherein the CDRs are derived from a non-human source and the remaining portion of the Ig molecule or fragment thereof is derived from a human antibody, preferably produced from a nucleic acid sequence encoding a human antibody
  • human antibody and “humanized antibody” are used herein to describe an antibody of which all portions of the antibody molecule are derived from a nucleic acid sequence encoding a human antibody Such human antibodies are most desirable for use in antibody therapies, as such antibodies would elicit little or no immune response in the human patient
  • chime ⁇ c antibody is used herein to describe an antibody molecule as well as antibody fragments, as described above in the definition of the term “humanized antibody "
  • the term “chime ⁇ c antibody” encompasses humanized antibodies Chime ⁇ c antibodies have at least one portion of a heavy or light chain ammo acid sequence derived from a first mammalian species and another portion of the heavy or light chain ammo acid sequence derived from a second, different mammalian species
  • the variable region is derived from a non-human mammalian species and the constant region is derived from a human species
  • the chime ⁇ c antibody is preferably produced from a nucleotide sequence from a non-human mammal encoding a variable region and a nucleotide sequence from a human encoding a constant region of an antibody
  • binds specifically is meant high avidity and/or high affinity binding of an antibody to a specific polypeptide Antibody binding to its epitope on a specific polypeptide is stronger than binding of the same antibody to any other epitope, particularly those which may be present in molecules in association with, or in the same sample, as the specific polypeptide of interest
  • Antibodies that bind specifically to a polypeptide of interest may be capable of binding other polypeptides at a weak, yet detectable, level, e g 10% or less of the binding shown to the polypeptide of interest Such weak binding, or background binding, is readily discernible from the specific antibody binding to the compound or polypeptide of interest, e g by use of appropriate controls
  • detectable labeled antibody detectably labeled anti-EGF
  • detectably labeled anti-EGF fragment is meant an antibody (or antibody fragment that retains binding specificity), having an attached detectable label The detectable label is normally attached by chemical conjugation, but where the label is a polypeptide
  • Detectable labels may be selected from a variety of such labels known in the art, but normally are radioisotopes, fluorophores, enzymes, e g horseradish peroxidase, or other moieties or compounds that either emit a detectable signal (e g radioactivity, fluorescence, color) or emit a detectable signal after exposure of the label to its substrate
  • Various detectable label/substrate pairs e g horseradish peroxidase/diaminobenzidme, avidin/streptavidm, luciferase/lucifenn
  • methods for labeling antibodies and methods for using labeled antibodies to detect an antigen are well known in the art (for example, see Harlow and Lane, eds (Antibodies A Laboratory Manual (1988) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) THERAPEUTIC METHODS
  • the present invention provides a method of treating pulmonary hypersecretion by administering therapeutic amounts of EGF-R antagonists
  • Any disease and particularly and pulmonary disease characterized by hypersecretion of mucus or accumulation of pathological levels of mucus may be treated by the methods described herein
  • pulmonary hypersecretory diseases that may be treated by this method include, but are not limited to, chronic obstructive lung diseases, such as chronic bronchitis, inflammatory diseases such as asthma, bronchiectasis, pulmonary fibrosis, COPD, diseases of nasal hypersecretion, e g nasal allergies, and other hypersecretory diseases
  • Genetic diseases such as cystic fibrosis, Kartagener syndrome, alpha-1-ant ⁇ trypsm deficiency, familial non-cystic fibrosis mucus spissation of respiratory tract, are intended to be included as well
  • Antagonists that directly target EGF or EGF-R are preferred
  • any factor or cell involved in the biological cascade that results in EGF-R promoting goblet cell proliferation may be targeted for inhibition, e g TGF- ⁇ antagonists
  • a cascade begins during an inflammatory response when cells such as mast cells or neutrophils release TNF- ⁇ , which then promotes EGF-R expression Stimulation of EGF-R, e g by its ligand EGF, in turn triggers goblet cell proliferation
  • any cells or factors involved in the cascade, such as in the TNF- ⁇ pathway may be targeted for antagonist activity
  • the EGF-R antagonist administered in the therapeutic method may be in any form
  • the EGF-R antagonist may be in the form of a small molecule (/ e , antisense ohgonucleotide, tyrosine kinase inhibitor, efc ), antibodies or portion of antibodies that bind to EGF, TGF ⁇ or EGF-R
  • SMALL MOLECULE EGF-R ANTAGONISTS Tyrosine kinase inhibitors that act on the EGF receptor, and that are selective for the EGF-R, are known in the art, and may be used in the subject methods Examples are described above, and of such may include BIBX1522 (Boeh ⁇ nger Ingelheim, Inc , Ingelheim, Germany), CGP59326B (Novartis Corporation, Basel, Switzerland), 4-am ⁇ noqu ⁇ nazohne EGF-R inhibitors (described in U S Patent no 5,760,041), substituted styrene compounds which can also be a naphthalene, an mdane or a benzoxazine, including nitnle and molononit ⁇ le compounds (described in U S Patent no 5,217,999), the inhibitors disclosed in U S Patent no 5,773,476, potato carboxypeptidase inhibitor (PCI), a 39-am ⁇ no acid protease inhibitor with three disul
  • Preferred tyrosine kinase inhibitors are selective for EGF receptor, / e the EGF-R is inhibited to a greater degree than other cell surface receptors having tyrosine kinase activity Selectivity is enhanced by the methods of formulation and drug delivery, e g where the inhibitor is preferentially delivered to inflamed airways, etc
  • Typical dosages for systemic administration range from 0 1 ⁇ g to 100 milligrams per kg weight of subject per administration Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects Some of the specific compounds are more potent than others Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means A preferred means is to measure the physiological potency of a given compound, for example with the in vitro and in vivo tests described herein
  • Antibodies as EGF-R antagonists are of particular interest (e g Vilona, ef al , American Journal of Pathology 151 1523)
  • Antibodies to EGF or EGF-R are produced by immunizing a xenogeneic immunocompetent mammalian host, including murine, rodentia, lagomorpha, ovine, porcine, bovine, efc with EGF or EGF-R or portions thereof
  • a xenogeneic immunocompetent mammalian host including murine, rodentia, lagomorpha, ovine, porcine, bovine, efc with EGF or EGF-R or portions thereof
  • human EGF or EGF-R or portions thereof are used as the immunogen
  • the choice of a particular host is primarily one of convenience
  • Immunizations are performed in accordance with conventional techniques, where the immunogen may be injected subcutaneously, intramuscularly, intrapentoneally, travascularly, efc into the host animal
  • EGF or EGF-R intrapentoneally every other day will be used as an immunogen
  • the injections may be with or without adjuvant, e g complete or incomplete Freund's adjuvant, specol, alum, efc
  • the antiserum may be harvested in accordance with conventional ways to provide polyclonal antisera specific for EGF or the EGF-R
  • Either monoclonal or polyclonal antibodies, preferably monoclonal antibodies, are produced from the immunized animal
  • Polyclonal antisera may be harvested from serum by conventional methods from the animals after completion of the immunization schedule
  • lymphocytes are harvested from the appropriate lymphoid tissue, e g spleen, draining lymph node, efc , and fused with an appropriate fusion partner, usually a myeloma line, producing a hybndoma secreting a specific monoclonal antibody
  • Screening clones of hybndomas for the antigenic specificity of interest is performed in accordance with conventional methods
  • EGF so as to inhibit binding of EGF to EGF-R, e g an antibody that specifically binds to the extracellular domain of EGF-R thereby preventing binding of EGF
  • Such antibodies may be made by conventional methodology described above, or are commercially available.
  • Examples of antibodies that would function as an EGF antagonist include, but are not limited to, the neutralizing anti-EGF-R monoclonal antibody C225 (Kawamoto ef at (1983) Proc. Natl Acad. Sci. (USA) 80:1337-1341 ; Petit et al. (1997) J. Path. 151:1523-153, produced by ImClone Systems New York, NY) and the anti-EGF-R monoclonal antibody EMD55900 (also called Mab 425), (Merck, Darmstadt, Germany).
  • the subject antibodies may be produced as a single chain, instead of the normal multimeric structure.
  • Single chain antibodies are described in Jost ef al. (1994) J.B.C. 269:26267-73, and others.
  • DNA sequences encoding the variable region of the heavy chain and the variable region of the light chain are ligated to a spacer encoding at least about 4 amino acids of small neutral amino acids, including glycine and/or serine.
  • the protein encoded by this fusion allows assembly of a functional variable region that retains the specificity and affinity of the original antibody.
  • the humanized antibody may be the product of an animal having transgenic human immunoglobulin constant region genes (see for example, International Patent Applications WO 90/10077 and WO 90/04036).
  • the antibody of interest may be engineered by recombinant DNA techniques to substitute the CH1 , CH2, CH3, hinge domains, and/or the framework residues with the corresponding human sequence (see WO 92/02190).
  • Ig cDNA for construction of chimeric immunoglobulin genes is also known in the art (Liu et al. (1987) P.N.A.S. 84:3439 and (1987) J. Immunol. 139:3521 ).
  • mRNA is isolated from a hybridoma or other cell producing the antibody and used to produce cDNA.
  • the cDNA of interest may be amplified by the polymerase chain reaction using specific primers (U.S. Patent nos. 4,683,195 and 4,683,202).
  • a library is made and screened to isolate the sequence of interest.
  • the DNA sequence encoding the variable region of the antibody is then fused to human constant region sequences.
  • human constant regions genes may be found in Kabat ef al. (1991) Sequences of Proteins of Immunological Interest, N.I.H. publication no. 91-3242. Human C region genes are readily available from known clones. The chimeric, humanized antibody is then expressed by conventional methods.
  • Antibody fragments such as Fv, F(ab') 2 and Fab may be prepared by cleavage of the intact protein, e.g. by protease or chemical cleavage.
  • a truncated gene is designed.
  • a chimeric gene encoding a portion of the F(ab') 2 fragment would include DNA sequences encoding the CH1 domain and hinge region of the H chain, followed by a translational stop codon to yield the truncated molecule.
  • An individual having a hypersecretory mucus disease may initially be administered amounts of EGF-R antagonist in the range of about 20 milligrams (mg) to about 400 mg per kilogram weight of patient twice daily, e.g. by inhalation.
  • EGF-R antagonist in the range of about 20 milligrams (mg) to about 400 mg per kilogram weight of patient twice daily, e.g. by inhalation.
  • the subject therapeutic agents are antisense molecules specific for human sequences coding for EGF or EGF-R
  • the administered therapeutic agent may be antisense ohgonucleotides, particularly synthetic ohgonucleotides having chemical modifications from native nucleic acids, or nucleic acid constructs that express such anti-sense molecules as RNA
  • the antisense sequence is complementary to the mRNA of the targeted EGF or EGF-R genes, and inhibits expression of the targeted gene products (see e g Nyce ef al (1997) Nature 385 720)
  • Antisense molecules inhibit gene expression by reducing the amount of mRNA available for translation, through activation of RNAse H or ste ⁇ c hindrance
  • One or a combination of antisense molecules may be administered, where a combination may comprise multiple different sequences from a single targeted gene, or sequences that complement several different genes
  • a preferred target gene is EGF-R or EGF
  • the gene sequence may be accessed through public databases (human epidermal growth factor, Genbank accession no K01166, human mRNA for precursor of epidermal growth factor receptor, Genbank accession no X00588) Generally, the antisense sequence will have the same species of origin as the animal host
  • Antisense molecules may be produced by expression of all or a part of the target gene sequence in an appropriate vector, where the vector is introduced and expressed in the targeted cells
  • the transcnptional initiation will be oriented such that the antisense strand is produced as an RNA molecule
  • the anti-sense RNA hybridizes with the endogenous sense strand mRNA, thereby blocking expression of the targeted gene
  • the native transcnptional initiation region, or an exogenous transcnptional initiation region may be employed
  • the promoter may be introduced by recombinant methods in vitro, or as the result of homologous integration of the sequence into a chromosome
  • Many strong promoters that are active in muscle cells are known in the art, including the ⁇ -actm promoter, SV40 early and late promoters, human cytomegaiovirus promoter, retroviral LTRs, efc Transcription vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucle
  • the antisense molecule is a synthetic o gonucleotide
  • Antisense ohgonucleotides will generally be from about 7 to 500, usually from about 12 to 50 nucleotides, more usually from about 20 to 35 nucleotides, where the length is governed by efficiency of inhibition, specificity, including absence of cross-reactivity, and the like It has been found that short ohgonucleotides, of from 7 to 8 bases in length, can be strong and selective inhibitors of gene expression (see Wagner ef al (1996) Nature Biotechnology 14 840-844)
  • a specific region or regions of the endogenous sense strand mRNA sequence is chosen to be complemented by the antisense sequence It has been shown that the 5' region of mRNA is particularly susceptible to antisense inhibition
  • Selection of a specific sequence for the ohgonucleotide may use an empirical method, where several candidate sequences are assa
  • Antisense ohgonucleotides may be chemically synthesized by methods known in the art (see Wagner ef al (1993) supra and Mil gan ef a/ , supra ) Preferred ohgonucleotides are chemically modified from the native phosphodiester structure, in order to increase their mtracellular stability and binding affinity A number of such modifications have been described in the literature, which alter the chemistry of the backbone, sugars or heterocyclic bases
  • Ohgonucleotides may additionally comprise a targeting moiety that enhances uptake of the molecule by cells
  • the targeting moiety is a specific binding molecules, e g an antibody or fragment thereof that recognizes molecules present on the surface of lung epithelial cells, particularly epithelial cells containing EGF-R
  • Bispecific antibodies, chimeric antibodies and single chain antibodies are known in the art
  • non-human antibodies can be humanized in various ways
  • Linkage between the ohgonucleotide and targeting moiety may use any conventional method, for example by disulfide, amide or thioether bonds, depending on the chemistry of the ohgonucleotide backbone
  • the linkage will be cleaved inside the cell to liberate the ohgonucleotide
  • Ohgonucleotides can be conjugated to hydrophobic residues, e g cholesterol, to protect from nucleases and to improve transport across cell membranes Alternatively, conjugation to poly-L-lysine or other polyammes may also enhance delivery to the cell A further modification that can be made is the addition of an intercalating component, such as acndine, capable of intercalating into the target mRNA and stabilizing the resultant hybrid Antisense ohgonucleotides may be transfected in combination with an enzyme(s) that will degrade antisense-mRNA complexes in the cell, e g RNase-H Any protein or enzyme that can preferentially degrade or sequester the antisense-mRNA duplex may be similarly useful
  • catalytic nucleic acid compounds e g ribozymes, anti-sense conjugates, etc may be used to inhibit gene expression
  • Ribozymes may be synthesized in vitro and administered to the patient, or may be encoded on an expression vector, from which the ribozyme is synthesized in the targeted cell (for example, see International patent application WO 9523225, and Beigelman et al (1995) Nucl Acids Res 234434-42)
  • Examples of ohgonucleotides with catalytic activity are described in WO 9506764
  • Conjugates of anti-sense ohgonucleotides with a metal complex, e g terpy ⁇ dylCu(ll) capable of mediating mRNA hydrolysis are described in Bashkm ef al (1995) Appl Biochem Biotechnol 54 43-56
  • EGF-R antagonists may be provided in solution or in any other pharmacologically suitable form for administration, such as a hposome suspension
  • the appropriate antibodies or other form of anti- EGF are formulated for administration in a manner customary for administration of such materials Typical formulations are those provided in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Company, Easton, PA
  • the route of administration will be selected based on the compound being administered, the status of the patient and disease that is being treated Where there is hypersecretion of mucus, a compound may be administered through different routes depending on the severity of the disease, e g emergency situations may require i v administration, acute but not life threatening situation may be treated orally, while chronic treatment can be administered by aerosol
  • the EGF antagonist may be administered by injection, including intramuscular, intravenous (IV), subcutaneous or peritoneal injection, most preferably IV and local injections
  • IV intravenous
  • other modes of administration may also be used provided means are available to permit the EGF-R antagonist to enter the systemic circulation, such as transmucosal or transdermal formulations, which can be applied as suppositories, skin patches, or mtranasally
  • Any suitable formulation that effects the transfer of the EGF-R antagonist to the bloodstream or locally to the lungs may properly be used
  • suitable formulations generally comprise aqueous solutions or suspensions using physiological saline, Hank's solution, or other buffers optionally including stabilizing agents or other minor components
  • suitable formulations can also be used
  • the EGF-R antagonist may also be supplied in lyophilized form and reconstituted for administration
  • Transmucosal and transdermal administrations generally include agents that facilitate passage through the mucosal or dermal barrier, such as bile, salts, fusidic acid and its analogs, various detergents and the like
  • suitable enteric coatings are formulated to permit the EGF-R antagonist to survive the digestive tract
  • the nature of the formulation will depend to some extent on the nature of the EGF-R antagonist chosen
  • a suitable formulation is prepared using known techniques and principles of formulation well known to those skilled in the art
  • the percentage of EGF-R antagonists contained in a particular pharmaceutical composition will also depend on the nature of the formulation, the percentage of an EGF-R antagonist that is an antibody will typically vary over a wide range from about 1 % by weight to about 85% by weight
  • Useful delivery systems include Sendai virus-liposome delivery systems (Rapaport and Shai
  • liposomes as a delivery vehicle is one method of interest for use with EGF-R antagonists.
  • the liposomes fuse with the cells of the target site and deliver the contents of the lumen intracellularly.
  • the liposomes are maintained in contact with the cells for sufficient time for fusion, using various means to maintain contact, such as isolation, binding agents, and the like.
  • Liposomes may be prepared with purified proteins or peptides that mediate fusion of membranes, such as Sendai virus or influenza virus, etc.
  • the lipids may be any useful combination of known liposome forming lipids, including cationic lipids, such as phosphatidylcholine.
  • the remaining lipid will normally be neutral lipids, such as cholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like.
  • neutral lipids such as cholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like.
  • the procedure described by Kato et al. (1991) J. Biol. Chem. 266:3361 may be used.
  • the EGF-R antagonist is encapsulated in a sterically stabilized
  • stealth liposomes e.g. pegylated liposomes.
  • liposomes When such liposomes are injected i.v., they remain in the circulation for long periods.
  • Postcapillary venular gap junctions open during airway inflammation and allow fluid accumulation and permit molecules, e.g. complement, kininogen, to enter tissues, initiating inflammatory cascades.
  • Such inflammation allows liposomes and their contents to be deposited selectively in the inflamed tissue (Zhang ef al. (1998) Pharm Res 15:455-460).
  • EGF-R antagonists may be administered to the afflicted patient by means of a pharmaceutical delivery system for the inhalation route.
  • the compounds may be formulated in a form suitable for administration by inhalation.
  • the pharmaceutical delivery system is one that is suitable for respiratory therapy by topical administration of EGF-R antagonists thereof to mucosal linings of the bronchi.
  • This invention can utilize a system that depends on the power of a compressed gas to expel the EGF-R antagonists from a container. An aerosol or pressurized package can be employed for this purpose.
  • aerosol is used in its conventional sense as referring to very fine liquid or solid particles carries by a propellant gas under pressure to a site of therapeutic application.
  • the aerosol contains the therapeutically active compound, which can be dissolved, suspended, or emulsified in a mixture of a fluid carrier and a propellant.
  • the aerosol can be in the form of a solution, suspension, emulsion, powder, or semi-solid preparation. Aerosols employed in the present invention are intended for administration as fine, solid particles or as liquid mists via the respiratory tract of a patient.
  • propellants include, but is not limited to, hydrocarbons or other suitable gas.
  • the dosage unit may be determined by providing a value to deliver a metered amount.
  • the present invention can also be carried out with a nebulizer, which is an instrument that generates very fine liquid particles of substantially uniform size in a gas
  • a nebulizer which is an instrument that generates very fine liquid particles of substantially uniform size in a gas
  • a liquid containing the EGF-R antagonists is dispersed as droplets
  • the small droplets can be carried by a current of air through an outlet tube of the nebulizer The resulting mist penetrates into the respiratory tract of the patient
  • a powder composition containing EGF-R antagonists or analogs thereof, with or without a lubricant, carrier, or propellant, can be administered to a mammal in need of therapy
  • This embodiment of the invention can be carried out with a conventional device for administering a powder pharmaceutical composition by inhalation
  • a powder mixture of the compound and a suitable powder base such as lactose or starch may be presented in unit dosage form in for example capsular or cartridges, e g gelatin, or blister packs, from which the powder may be administered with the aid of an inhaler
  • Combination therapies may be used to treat hypersecretory pulmonary disease
  • EGF-R antagonists may be combined with conventional treatment for alleviation of hypersecretion, such as bronchiodilators, corticosteroids, expectorants, mucolytic agents and the like to facilitate mucociliary clearance
  • a formulation of the present invention by injection (e g , intravenous) or by inhalation Patients which have large amounts of mucus in the lungs cannot, in general, be treated initially by inhalation This is due to the fact that the patient's lungs are sufficiently obstructed that inhaling aerosolized formulation into the lungs may not be particularly effective
  • administration by inhalation is preferred
  • Administration by inhalation is preferred because smaller doses can be delivered locally to the specific cells which are most in need of treatment By delivering smaller doses, any adverse side effects are eliminated or substantially reduced By delivering directly to the cells which are most in need of treatment, the effect of the treatment will be realized more quickly
  • Antagonists of the present invention can be formulated in basically three different types of formulations for inhalation
  • antagonists of the invention can be formulated with low boiling point propellants
  • Such formulations are generally administered by conventional meter dose inhalers (MDI's)
  • MDI's can be modified so as to increase the ability to obtain repeatable dosing by utilizing technology which measures the spiratory volume and flow rate of the patient as discussed within U S Patents 5,404,871 and 5,542,410
  • the agonists of the present invention can be formulated in aqueous or ethanohc solutions and delivered by conventional nebulizers
  • solution formulations are aerosolized using devices and systems such as disclosed within U S Patent 5,497,763, 5,544,646, 5,718,222, and 5,660,166
  • agonist compounds of the present invention can be formulated into dry powder formulations Such formulations can be administered by simply inhaling the dry powder formulation after creating an aerosol mist of the powder Technology for carrying such out is described within U S Patent 5,775,320 issued July 7, 1998 and U S Patent 5,740,794 issued April 21 , 1998
  • U S Patent 5,775,320 issued July 7, 1998 U S Patent 5,740,794 issued April 21 , 1998
  • applicants point out that these patents cite other publications in intrapulmonary drug delivery and such publications can be referred to for specific methodology, devices and formulations which could be used in connection with the delivery of agonists of the present invention
  • each of the patents are incorporated herein by reference in their entirety for purposes of disc
  • Screening assays may be used to identify bioactive candidate agents that are EGF antagonists Of particular interest are screening assays for agents that have a low toxicity for human cells
  • assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, and the like
  • the purified EGF or EGF-R protein may also be used for determination of three-dimensional crystal structure, which can be used for modeling intermolecular interactions, transporter function, etc
  • agent as used herein describes any molecule, e g protein or pharmaceutical, with the capability of altering or inhibiting the physiological function of EGF or EGF-R
  • a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations Typically, one of these concentrations serves as a negative control, / e at zero concentration or below the level of detection
  • Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized ohgonucleotides and ohgopeptides Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, este ⁇ fication, amidification, efc to produce structural analogs
  • the screening assay is a binding assay
  • the label can directly or indirectly provide a detectable signal
  • Various labels include radioisotopes, fluorescers, chemiluminescers, enzymes, specific binding molecules, particles, e g magnetic particles, and the like
  • Specific binding molecules include pairs, such as biotin and streptavidm, digoxin and antidigoxin, etc
  • the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures
  • reagents may be included in the screening assay These include reagents like salts, neutral proteins, e g albumin, detergents, etc that are used to facilitate optimal protem- protein binding and/or reduce non-specific or background interactions Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc may be used The mixture of components are added in any order that provides for the requisite binding
  • Incubations are performed at any suitable temperature, typically between 4 and 40° C Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening Typically between 0 1 and 1 hours will be sufficient
  • the compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host for treatment of hypersecretory disease in formulations described herein Depending upon the manner of introduction, the compounds may be formulated in a variety of ways described herein
  • the concentration of therapeutically active compound in the formulation may vary from about 0 1-100% by weight
  • the appropriate dosage level will also vary depending on a number of factors including the nature of the subject to be treated, the particular nature of the hypersecretory condition to be treated and its severity, the nature of the EFGR antagonist used as active ingredient, the mode of administration, the formulation, and the judgement of the practitioner For example, when antibodies are administered by themselves such as anti-EGF or EGF-R in an mjectable formulation, the dosages will be in the range of 20 mg/kg to about 40 mg/kg at a single dosage Repeated administration over a period of days may be required or administration by intravenous means may be continuous For chronic conditions, administration may be continued for longer periods as necessary
  • Efficacy of the dosing regime will be determined by assessing for improved lung function in the patient This assessment may include viscoelasticity measurements of sputum, improvements in pulmonary function, including improvements in forced exploratory volume of sputum and maximal midexpiratory flow rate
  • the aforementioned therapeutic regime can be given in conjunction with adjunct therapies such as antibiotics, DNAse I or other current therapies for the treatment of hypersecretory pulmonary disease If antibiotics are co-administered as part of the patient's therapy, bacterial quantitation following therapy can be included to assess the efficacy of the treatment by decreased bacterial growth, indicating decreased viscosity of mucus or sputum and increase of the mucus or sputum lung clearance
  • Pulmonary function tests as well as diagnostic tests for the clinical progression of pulmonary hypersecretory disease, are known to those individuals with skill in this art
  • Standard pulmonary function tests include airway resistance (AR), forced vital capacity (FVC), forced expiratory volume in 1 second (FEV(1)), forced midexpiratory flow, and peak expiratory flow rate (PEFR)
  • Other pulmonary function tests include blood gas analysis, responses to medication, challenge and exercise testing, measurements of respiratory muscle strength, fibro-optic airway examination, and the like
  • Some basic procedures for studying the properties of mucus include rheology, e g with the use of a magnetic microrheometer, adhesivity to characterize the forces of attraction between an adherent surface and an adhesive system by measuring the contact angle between a mucus drop and a surface Mucus transport by cilia can be studied using conventional techniques, as well as direct measurement, / e in situ mucus clearance Transepithehal potential difference, the net result of the activity of the ion-transport system of the pulmonary epithel
  • the patient to be treated can be a primate, such as a human, or any other animal exhibiting the described symptoms While the method of the invention is especially adapted for the treatment of a human patient, it will be understood that the invention is also applicable to veterinary practice
  • in vitro assays are used to assess the therapeutic potential of candidate agents to inhibit goblet cell proliferation, / e whether such agents are active as an EGF antagonist
  • such assays will comprise the following steps (i) contacting an in vitro model of goblet cell proliferation with EGF or the functional equivalent thereof, (n) subsequently contacting the in vitro model with a candidate agent, and (in) assessing goblet cell proliferation, wherein an inhibition of goblet cell proliferation is indicative of the candidate agent's therapeutic potential
  • rat tracheal cells can be isolated and maintained in culture as described in Guzman et al (1995) 217 412-419 Briefly, the rat tracheal cells are plated onto collagen gel coated semipermeable membranes, initially cultured submerged in media, and subsequently maintained with an air/hquid interface
  • in vitro cells include primary human bronchial cells (available from Clonetics, San Diego), NCI-H292 cells (ATCC CRL-1848), and A431 cells (ATCC CRL-1555)
  • the in vitro culture is contacted with EGF and with a candidate agent
  • the candidate agent may be contacted with the culture prior to, concurrently with, or subsequently to the addition of EGF depending on the endpoint to be assessed and the nature of the candidate agent
  • the cultured cells are assessed for inhibition of goblet cell proliferation relative to controls
  • molecular or biochemical markers may be used to assess goblet cell proliferation
  • molecular or biochemical markers include, but are not limited to, gene expression or protein expression characteristic of goblet cells
  • Certain mucin genes e g MUC5B (Desseyn et al (1997) J Biol Chem 272 3168-3178) are expressed in the airway, and have a gene product highly represented in mucus Expression of mucin genes provides a suitable marker for determining production of mucus Mucin gene expression may be assessed by conventional technology such as northern blot analysis, polymerase chain reaction (PCR), examination of the mucin gene promoter, or in situ analysis Alternatively mucin proteins are assessed by conventional methodology, such as western blot analysis, ELISA, immunochemistry and the like, using detectably labeled antibodies Morphological criteria may also be used to determine the presence or absence of goblet cells in the culture, such as staining for mucins using Alcian blue/PAS staining (Lou et)
  • in vivo animal models are used to assess the therapeutic potential of candidate agents to inhibit goblet cell proliferation
  • the assay comprises the steps of (i) creating an animal model of hypersecretory pulmonary disease by inducing EGF-R expression, ( ) stimulating the induced EGF-R to produce mucin producing goblet cells, (in) treating with a candidate agent, and (iv) assessing goblet cell proliferation or mucus secretion, wherein an inhibition of goblet cell proliferation or mucus secretion is indicative of the candidate agent's therapeutic potential
  • Any in vivo model of hypersecretory pulmonary disease may be used by way of example an asthmatic mouse model, as described in Temann ef al (1997) Am J Respir Cell Biol 16 471-478, and as shown in the examples provided herein Alternatively, a rat model can be used, as described by Takeyama ef al (1998) Am J Physiol Examples of other animal models that may be used include, but are not limited to Guinea pigs (a species that expresses goblet cells constitutively) and rats
  • the lung tissue or tracheal tissue of the animal models may be assessed by the same molecular and biochemical markers described for the in vitro model A decrease in goblet cell proliferation is indicative of the therapeutic potential of the EGF-R antagonist
  • Goblet cell hyperplasia occurs in various hypersecretory diseases of airways, but because the underlying mechanisms are unknown, no effective therapy exists In healthy airways, goblet cells are few, but in hypersecretory airway diseases, goblet cell hyperplasia occurs A human bronchial
  • NCI-H292 tumor necrosis factor alpha
  • instillation of TNF ⁇ , followed by EGF-R hgands resulted in an increased number of goblet and pre-goblet cells and a striking increase in Alcian blue/PAS-positive staining (reflecting mucous glycoconjugates) and mucin MUC5 gene expression
  • ovalbumin resulted in goblet cell production and EGF-R expression in airway epithelium
  • NCI-H292 cells in rats stimulated by TNF ⁇ followed by EGF-R hgands, and in the asthma model in rats, pretreatment with EGF-R tyrosine kinase inhibitor (BI)
  • EGF fetal bovine serum
  • penicillin 100 U/ml
  • streptomycin 100 ⁇ g/ml
  • cells were incubated with EGF (recombinant human EGF, 25 ng/ml, Genzyme, Cambridge, MA), TGF ⁇ (recombinant human TGF ⁇ , 25 ng/ml, Genzyme), TNF ⁇ (recombinant human TNF ⁇ , 20ng/ml, Genzyme), EGF (25 ng/ml) plus TNF ⁇ (20ng/ml) or TGF ⁇ (25 ng/ml) plus TNF ⁇ (20 ng/ml) for 12 h, 24 h or 48 h
  • EGF-R tyrosine kinase inhibitor BIBX1522 (10 ⁇
  • Triton X 1% sodium dioxycolate and PMSF (10 mg/ml) Total amount of protein was estimated by BCA protein assay reagent (Pierce, Rockford, IL) Cell lysates were boiled with Tricme sample buffer and 2% ⁇ ME at 95°C Proteins were separated by SDS-PAGE in 8% acrylamide gels The resulting gels were equilibrated in the transfer buffer 25 mM T ⁇ s-HCI, 192 mM glycme, 20% (vol/vol) methanol, pH 8 3 The proteins were then transferred electrophoretically to nitrocellulose membranes The membranes were then incubated for 1 h in 5% fat-free skim milk in PBS containing 0 05% Tween 20 Then the membranes were incubated with monoclonal mouse anti-EGF-R antibody (1 100) at 4°C overnight Bound antibody was visualized according to standard protocols for avidin-biotin-alkahne phosphatase complex method (ABC kit, Vector
  • Probes EGF-R mRNA expression was determined using the linearized pTRI-EGF-R-human probe template (Ambion, Austin, TX) This probe contains a 360 bp cDNA fragment of the human EGF-R gene, which spans exons 12-14.
  • MUC5 gene expression was determined using human MUC5AC probe, which contains a 298 bp cDNA fragment of human MUC5AC gene (generously provided by Dr. Carol Basbaum).
  • Hybridization solution contained 250 mM Tris-HCl (pH7.5), 5% SDS, 1 % BSA, 1% polyvinyl-pyrrolidone, 1% Ficoll, and 0.5% sodium pyrophosphate. After hybridization, the membranes were washed twice with 2 x SSC with 0.1% SDS for 30 min at room temperature, followed by two washes in 2 x SSC with 0.1% SDS for 30 min at 50°C and a rinse in 0.1 x SSC with 0.1% SDS. Membranes were exposed to X-ray film.
  • EGF 600 ng, 100 ⁇ l
  • TGF ⁇ rat synthetic TGF ⁇ , 250 ng, 100 ⁇ l; Sigma, St Louis, Ml
  • TNF ⁇ 200 ng, 100 ⁇ l
  • sterile PBS 100 ⁇ l was instilled into the trachea as control.
  • EGF-R tyrosine kinase inhibitor BIBX1522 (dose estimated from studies using the inhibitor to prevent cancer growth). Rats were pretreated with BIBX1522 (3, 10 or 30 mg/kg, i.p.), 1 h before and 24 h after instillation of TGF ⁇ . The trachea and lungs were removed for examination 48 h after the instillation of TGF ⁇ .
  • Rats were sensitized on days 0 and 10 with intraperitoneal injections of ovalbumin (10 mg, grade V; Sigma, St. Louis, MO), complexed with 100 mg of aluminum hydroxide in 0.5 ml of sterile saline. Rats then rested for 10 days. On day 20, ovalbumin was delivered directly into the trachea; animals were challenged with 100 ⁇ l of 0.1% ovalbumin in saline by intratracheal instillation three times (days 20, 22 and 24). Rats were euthanized either without challenge (day 20), or 48 h after the third challenge (day 26). This procedure induced goblet cell metaplasia.
  • sensitized rats were pretreated with an EGF-R tyrosine kinase inhibitor, BIBX1522.
  • BIBX1522 10 mg/kg, i.p., 1 h before the challenge
  • BIBX1522 was also instilled into the trachea together with ovalbumin (BIBX1522, 10 _5 M, 100 ⁇ l).
  • BIBX1522 was also injected i.p. every 24 h until the day before the rats were euthanized. After the animals were euthanized, the trachea was removed 48 h after the third challenge.
  • Tissue preparation At preselected times during anesthesia, the systemic circulation was perfused with 1% paraformaldehyde in DEPC-treated PBS at a pressure of 120 mmHg. The trachea was then removed and placed in 4% paraformaldehyde for 24 h. After fixation, trachea and lungs were embedded in either JB-4 plus monomer solution A for cell analysis or O.C.T. compound (Sakura Finetek U.S.A., Inc., Torrance, CA) for immunohistochemistry and in situ hybridization. The embedded tissues were cut as cross sections (4 mm thick) and placed on slides.
  • Goblet cells are tall, cuboidal, goblet to low columnar in shape, with abundant Alcian blue/PAS-stained granules filling most of the cytoplasm between the nucleus and the luminal surface.
  • Pre-goblet cells are defined as cells with smaller mucus-stained areas ( ⁇ 1/3 height in epithelium from basement membrane to luminal surface) or with sparsely and lightly Alcian blue/PAS-stained, small granules.
  • Ciliated cells are recognized by their ciliated borders, lightly stained cytoplasm, and large round nuclei.
  • Non-granulated secretory cells are columnar in shape and extend from the lumen to the basal lamina.
  • the cytoplasm stains light pink color, and a few tiny PAS-positive and Alcian blue-negative granules are observed in the cytoplasm.
  • Basal cells are small flattened cells with a large nucleus, located just above the basal lamina but not reaching the airway lumen. Quantification of goblet cell production. Goblet cell production, was determined by the volume density of Alcian blue/PAS-stained mucosubstances on the mucosal surface epithelium using a semi-automatic imaging system described elsewhere (Weber ef al. (1984) Science 224:294-297).
  • EGF-R Immunohistochemical localization of EGF-R in rat epithelium
  • the localization of EGF-R was examined using immunohistochemical stammg with an antibody to EGF-R (Calbiochem, San Diego, CA) in frozen sections of rat trachea
  • tissues were placed in 4% paraformaldehyde in PBS for 1 h and then removed in 30% sucrose for cryoprotection overnight Tracheas were embedded in O C T compound (Sakura Finetek U S A , Inc , Torrance, CA) and frozen Frozen sections (5 ⁇ m) were cut and placed on glass slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA) Immunostaining was performed similarly to the in vitro studies
  • RNA probes for in situ hybridization A 320 bp cDNA fragment of rat MUC5 was subcloned into the Xba/hmdlll site of the transcription vector, pBluescr ⁇ pt-SK(-) (Stratagene, La Jolla, CA)
  • this recombinant plasmid containing the rat MUC5 cDNA fragment was linearized and transcribed in vitro with the T7 or T3 polymerase to obtain antisense or sense probe, respectively
  • the probes for in situ hybridization were generated in the presence of [ 35 S]UTP
  • the cDNA template was digested with DNase, and radiolabeled RNA was purified via a Sephadex G-25 Quick SpinTM Column (Boehrmger Mannheim, Indianapolis, IN) and precipitated in an ethanol/ammonium acetate solution Before use, RNA probes were washed with 70% ethanol and diluted in 10 mM DTT
  • NCI-H292 cells express EGF-R constitutively.
  • Western analysis of immunoblots identified the presence of EGF-R protein in confluent cultures of NCI-H292 cells ( Figure 1A, right). Cells were examined after becoming confluent. Lysates were electrophoresed in 8% acrylamide gels and blotted with anti-EGF-R antibody. Molecular weights of marker proteins are reported on the right.
  • a positive control for EGF-R was protein from A431 cells ( Figure 1A, left), which express EGF-R constitutively (Weber et al., supra.).
  • Fig 1B Immunocytochemical analysis with anti-EGF-R antibody in cultures of NCI-H292 cells. At confluence, positive staining was seen, most strongly in dividing cells (arrows, right side). In the absence of the primary antibody, staining was absent (left side).
  • Northern blotting showed that TNF ⁇ (20 ng/ml) up-regulated EGF-R gene expression, an effect that was present at 12 h and increased at 24 h (Fig. 1C, Northern analysis of EGF-R in NCI-H292 cells).
  • EGF-R Ligands Stimulate Expression of Mucous Glycoconjugates and MUC5 Gene
  • EGF-R are expressed constitutively in NCI-H292 cells, so we assessed the ability of EGF-R ligands (EGF, TGF ⁇ ) to induce the production of mucous glycoconjugates (Figure
  • EGF-R tyrosine kinase inhibitor BIBX1522 (10 ⁇ g/ml). When cells were incubated alone (control), some PAS-positive staining was seen (arrows, upper column); incubation with TNF ⁇ (20 ng/ml) alone did not affect the staining; incubation with EGF; (25 ng/ml) or with TGF ⁇ (25 ng/ml) increased the
  • NCI-H292 cells showed some expression in the control state (Figure 3, lower left column), when the cells were incubated with EGF or TGF ⁇ , MUC5 gene expression was barely recognized at 12 h but was clearly expressed at 24 h TNF ⁇ alone did not affect MUC5 gene expression, but when TNF ⁇ was added to the cells incubated with EGF-R hgands, MUC5 gene expression increased markedly above the level caused the EGF-R hgand alone ( Figure 3)
  • EGF-R Tyrosine Kinase Inhibitor (BIBX1522) Prevents Expression of Mucous Glycoconjungates and of MUC5 Gene Expression in NCI-H292 Cells
  • BIBX1522 EGF-R tyrosine kinase inhibitor
  • FIG. 2 On Northern analysis, MUC5 gene expression that was markedly increased by the combination of TNF ⁇ plus EGF or plus TGF ⁇ was completely inhibited by pre-incubation with BIBX1522 ( Figure 3, lower column)
  • tracheal epithelium contained few EGF-R-positive cells ( Figure 4A, left)
  • intratracheal instillation of TNF ⁇ (200 ng) induced EGF-R protein in various cell types in the tracheal epithelium ( Figure 4A, right) EGF-R-positive staining was present in goblet cells (G), pre-goblet cells (P-G), non-granulated secretory cells (S), and basal cells (Ba), but not in ciliated cells
  • TNF ⁇ induces EGF-R protein production Role of EGF-R Ligands in Production of Mucous Glycoconjugates and MUC5 Gene Expression in Rats
  • tracheal epithelium contained few goblet and pre-goblet cells Intratracheal epithelium
  • EGF-R Tyrosine Kinase Inhibitor (BIBX1522) Prevents Goblet Cell Production Induced by
  • EGF-R when stimulated by EGF-R hgands, induce goblet cell production in vitro and in vivo, effects due to activation of EGF-R and which were blocked by an EGF-R tyrosine kinase inhibitor In an ovalbumin model of asthma, the inhibitor was also effective in preventing goblet cell production
  • Asthma serves as an example of the therapeutic strategy of the invention
  • Normal human airway epithelium has a ratio of 3-10 ciliated cells to each goblet cell
  • the number of goblet cells can be equal or exceed ciliated cells, in patients who die in sfafivs asthmaticus, there is a 30-fold increase in the percentage area occupied by goblet cells compared with the number in patients dying of non-asthma respiratory diseases
  • Inhibition of production of goblet cells should eliminate this source of hypersecretion
  • the life cycle of goblet cells is unknown, the time course of resolution of goblet cell hyperplasia with treatment can not be predicted with precision
  • Inhibition of EGF-R activation may inhibit goblet cell hyperplasia much more rapidly, depending on the life span of goblet cells
  • Hypersecretion is a major manifestation in many chronic inflammatory diseases of airways
  • Present findings provide a mechanism and a strategy for therapy by inhibiting EGF-R activation, goblet cell production is prevented
  • Inhibitors of EGF-R activation are proposed as therapy in hypersecretory airway diseases
  • Neutrophil isolation was performed by standard techniques of Ficoll-Hypaque gradient separation, dextran sedimentation, and ' hypotonic lysis of erythrocytes Cells were routinely
  • NCI-H292 cells a human pulmonary mucoepidermoid carcinoma cell line, were grown in RPMI 1640 medium containing 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 ⁇ g/ml) and Hepes (25 mM) at 37°C in a humidified 5% C0 2 water-jacketed incubator Either 6- well culture plates or 8-chamber slides were used to culture the cells When confluent, cells were incubated for 1 h with neutrophils (10 6 cells/ml) alone, TNF ⁇ alone (recombinant human TNF ⁇ , 20ng/ml, Genzyme, Cambridge, MA), IL-8 (recombinant human IL-8, 10 8 M, Genzyme) alone, fMLP (10 8 M, Sigma, St Louis, MO) alone, TNF ⁇ plus neutrophils, IL-8 plus neutrophils, fMLP plus neutrophils, hydrogen peroxide (H2O2, 200 ⁇ M), cigarette smoke solution or TGF
  • Cigarette smoke solution was prepared as previously described (Dusser et al (1989) J Clin Invest 84 900-906)
  • MUC5AC mouse mAb to MUC5AC (clone 45 M1 , 1 200, Neo Markers, Fremont, CA) for 1 h at room temperature, and then washed 3 times with PBS to remove excess primary antibody
  • TGF ⁇ protein was measured using a commercially available kit for ELISA (Sigma), following the manufacturer's instructions Supernatant taken after incubation of neutrophils plus TNF ⁇ (20 ng/ml) for 1 h was mixed with the lysis buffer PBS containing
  • Triton X-100 1% Triton X-100, 1% sodium deoxycholate and several protease inhibitors, (Complete Mini, Boehrmger Mannheim, Germany), and then used to measure TGF ⁇
  • EGF-R Tyrosine Kinase Inhibitors Prevent MUC5AC Synthesis Induced by Supernatant of Activated Neutrophils Because EGF-R hgands are known to cause MUC5AC synthesis in NCI-H292 cells via activation of EGF-R tyrosine kinase, the role of EGF-R activation in MUC5AC synthesis induced by the supernatant of activated neutrophils was examined Pretreatment of NCI-H292 cells with selective EGF-R tyrosine kinase inhibitors (BIBX1522, AG148), prevented the MUC5AC protein synthesis that was usually induced by the supernatant of activated neutrophils A selective platelet- derived growth factor receptor kinase inhibitor (AG1295) and a negative control for tyrphostins (A1) were without effect. These results implicate activation of EGF-R tyrosine kinase in MUC5AC synthesis induced by the
  • EGF-R tyrosine kinase is dependent on the EGF-R hgands (EGF and TGF ⁇ ).
  • EGF and TGF ⁇ EGF-R hgands
  • Pretreatment of the supernatant with either anti-TGF ⁇ antibody or anti- EGF antibody did not inhibit MUC5AC synthesis induced by the supernatant of activated neutrophils
  • TGF ⁇ was not detected in the supernatant
  • EGF-R tyrosine phosphorylation caused by the supernatant of activated neutrophils was induced by a mechanism independent of the EGF-R hgands, EGF and TGF ⁇
  • Cigarette smoke and the oxygen free radical, H 2 0 2 up-regulated MUC5AC gene expression within 12 h, as did TGF ⁇ Likewise, all stimuli increased MUC5AC protein synthesis and mucous glycoconjugate production within 24 h, effects that occurred in a dose-dependent fashion
  • the maximum MUC5AC synthesis in response to H 2 0 2 was significantly less than the response to TGF ⁇
  • Pretreatment with AG1478 prevented the increase in MUC5AC protein synthesis induced by all stimuli, indicating that the stimuli cause mucin synthesis by the activation of EGF-R tyrosine kinase MUC5AC synthesis by supernatant of activated neutrophils, cigarette smoke and H 2 0 2 were significantly inhibited by pretreatment with free radical scavengers (DMSO and DMTU) and SOD, but MUC5AC protein synthesis by TGF ⁇ was unaffected by DMSO or SOD In
  • Agarose plugs Agarose plugs (0 7-0 8 mm diameter) were made with 4% agarose type II medium EEO (Sigma, St Louis, MO) in sterile phosphate-buffered saline (PBS) To visualize the agarose plugs in tissue, 3% suspension Monastral blue B (Sigma, St Louis, MO) was added after melting the agarose at 50°C
  • mice were treated with BIBX1522 (80 mg/kg, i p ) 1 h before instillation of the agarose plugs and repeated daily (40 mg/kg, i p , bid) Animals were euthanized 24, 48 or 72 h after instillation of the agarose plugs.
  • animals were treated with a TNF ⁇ neutralizing antibody (Genzyme, Boston, MA) The first treatment (100 ⁇ l in 0 2 ml saline, i p ) was given 1 h before the instillation of agarose plugs, and i p injections were repeated daily
  • TNF ⁇ neutralizing antibody was infused (10 ⁇ l/h) via an osmotic minipump (Alzet 2ML1 , Alza Corp , Palo Alto, CA) implanted
  • All drugs (BIBX1522, TNF ⁇ neutralizing antibody, cyclophosphamide, and NPC 15669) were given i p 1 h before instillation of agarose plugs, and doses were repeated daily for 3 d Tissue preparation.
  • rats were anesthetized with sodium pentobarbital (65 mg/kg, i.p.), the systemic circulation was perfused with 1% paraformaldehyde in diethyl pyrocarbonate-treated PBS at a pressure of 120 mmHg.
  • the right lung was removed, and the right caudal lobe was used for histology.
  • tissues were removed and placed in 4% paraformaldehyde for 1 h and then replaced in 30% sucrose for cryoprotection overnight.
  • the tissues were embedded in O.C.T. compound (Sakura Finetek U.S.A., Inc., Torrance, CA).
  • methacrylate sections the tissues were placed in 4% paraformaldehyde for 24 h and then dehydrated with graded concentrations of ethanol and embedded in methacrylate JB-4 (Polysciences, Inc., Warrington, PA). Tissue sections (4 ⁇ m thick) were stained with Alcian blue/PAS and counterstained with hematoxylin.
  • Morphometric analysis of bronchial epithelium The percentage of Alcian blue/PAS-stained area of mucous glycoconjugates in the epithelium was determined using a semi-automatic image analysis system according to previously published methods. The area of epithelium and Alcian blue/PAS-stained mucous conjugates within the epithelium was manually circumscribed and analyzed using the NIH Image program (developed at the U.S. National Institutes of health and available from the internet by ananymous FTP or from a floppy disk from the National Technical Information Service, Springfield, Virginia; part number PB95-500195GEI). The data are expressed as the percentage of the total epithelial area occupied by Alcian blue/PAS stain.
  • the percentage of the length of epithelial surface occupied by Alcian blue/PAS-positive staining was determined by calculating the length that stained positively as a ratio to the total length.
  • the percentage of denuded epithelium was determined by calculating the ratio of the length of denuded epithelium to the total epithelial length.
  • the total number of epithelial cells was determined by counting epithelial cell nuclei over 2 mm of the basal lamina with an oil immersion objective lens (x1000 magnification).
  • the linear length of the basal lamina under each analyzed region of epithelium was determined by tracing the contour of the digitalized image of the basal lamina.
  • the epithelial cells were identified as described previously. In brief, basal cells were identified as small flattened cells with a large nucleus, located just above the basal lamina but not reaching the airway lumen. The cytoplasm stained darkly, and Alcian blue or PAS-positive granules were not present.
  • Ciliated cells were recognized by their ciliated borders, lightly stained cytoplasm, and large, round nucleus.
  • Non-granulated secretory cells were columnar in shape and extended from the bronchial lumen to the basal lamina. After intrabronchial instillation of agarose plugs, "developing" goblet cells (pre-goblet cells) were formed.
  • EGF-R Immunohistochemical localization of EGF-R The presence of EGF-R was determined by immunohistochemical localization, using a monoclonal mouse antibody to the EGF-R (Calbiochem, San Diego).
  • Biotinylated horse anti-mouse IgG (1 200, Vector Lab , Burhngame, CA), followed by streptavidin- peroxidase complex (ABC kit, Vector Lab , Burhngame, CA) was used to visualize antigen-antibody complexes stained with 3, 3'-d ⁇ am ⁇ nobenz ⁇ d ⁇ ne tetrahydrochlonde (Sigma, St Louis, MO) Negative control slides were incubated with either the primary or secondary antibody omitted and replaced with
  • Bronchoalveolar lavage(BAL) To assess differential cell counts in each group of animals, lungs were lavaged five times with 3 ml aliquots of sterile PBS, lavages were pooled, and the volume was measured Cells in BAL were collected by spinning the lavage fluid at 1,000 rpm for 10 m Ten microliters of a cell suspension was then counted with a hematocytometer to determine cell numbers in BAL fluid Differential cell counts were performed on cytospun preparations stained with Diff-Quik (American Scientific Products, McGaw Park, IL) Differential cell counts were obtained by sampling at least 200 cells on each cytospun slide
  • Effecf of agarose plugs on EGF-R expression in airway epithelium In control animals, immunostainmg with an antibody to EGF-R showed sparse staining in epithelium However, after instillation of agarose plugs, epithelium adjacent to agarose plugs showed EGF-R-positive staining in cells that stained positively with Alcian blue/PAS The staining pattern for EGF-R paralleled the staining for MUC5AC and AB/PAS Pre-goblet, goblet, and non-granulated secretory cells were immunopositive for EGF-R Ciliated cells showed no immunoreactivity In airways not obstructed by agarose plugs, the epithelium showed little staining for EGF-R and appeared similar to staining in control animals Effecf of EGF-R tyrosine kinase inhibitor on goblet cell metaplasia and on mucin gene expression.
  • EGF-R is a member of the class of tyrosine kinase receptors.
  • EGF-R ligands EGF or TGF ⁇
  • a specific EGF-R tyrosine kinase is activated. Therefore, to test the hypothesis that EGF-R activation induces expression of MUC5AC gene and of mucous glycoconjugates after instillation of agarose plugs, an EGF-R tyrosine kinase inhibitor (BIBX1522) was injected intrapentoneally in rats.
  • BIBX1522 markedly inhibited agarose plug-induced Alcian blue/PAS-stained area of epithelium at 24, 48 and 72 h. It also completely inhibited the expression of MUC5AC gene at 72 h after plug instillation.
  • Infiltrating cells were also evaluated in tissue sections: airways without agarose plugs contained few neutrophils, but airways containing plugs showed presence of neutrophils, both in the epithelium and in the lumen.
  • EGF-R are not normally expressed in airway epithelium of pathogen-free rats but is induced by TNF ⁇ .
  • EGF-R ligands EGF or TGF ⁇
  • a selective inhibitor of EGF-R tyrosine kinase (BIBX1522) completely inhibits these responses, implicating EGF-R signaling in goblet cell metaplasia.
  • BIBX1522 inhibited agarose plug-induced production of mucous glycoconjugates and MUC5AC gene expression. These results implicate an EGF-R cascade in agarose plug-induced goblet cell metaplasia.
  • Cyclophosphamide a drug that selectively depresses leukocyte production, prevented neutrophil recruitment into airway lavage fluid and into the airway epithelium following the instillation of agarose plugs and also prevented agarose plug-induced goblet cell metaplasia. Macrophages were also increased after the introduction of agarose plugs, but cyclophosphamide did not inhibit macrophage recruitment. These results implicate neutrophils in agarose plug-induced goblet cell metaplasia. The fact that cyclophosphamide also decreased EGF-R protein expression after agarose plugs suggests that neutrophils contribute, at least in part, to the EGF-R expression in this inflammatory condition.
  • Neutrophils are also capable of producing the EGF-R ligands, EGF and TGF ⁇ .
  • epithelial cells are sources of EGF-R ligands, and there was striking denudation of epithelium adjacent to the agarose plugs.
  • the epithelium could be an important potential source of both TNF ⁇ and EGF-R ligands.
  • Epithelial damage is a common finding in studies of patients even with mild asthma, and the damage is increasingly related to worsening of clinical symptoms.
  • Epithelial damage produced by the allergic response may induce EGF-R activation, which results in abnormal goblet cell production.
  • the data presented above implicate EGF-R activation in a different response, specifically involving goblet cell metaplasia.
  • Mechanical epithelial damage and epithelial injury in asthma may involve a similar (EGF-R) cascade, resulting in abnormal growth of epithelial secretory cells. This provides a mechanism for the hypersecretion that occurs in fatal cases of acute asthma.
  • Example 4 Regranulation of goblet cell by EGF- Receptors Degranulation of goblet cells in rat nasal respiratory epithelium was induced by infranasal inhalation of fMLP. Significant degranulation was induced in the nasal septal epithelium 4 h after infranasal inhalation of fMLP (10 "7 M). Goblet cell regranulation occurred by 48 h after inhalation. In the control state, MUC5AC protein was expressed in the goblet cells, but EGF-R protein was not expressed. Both EGF-R and MUC5AC mucin gene and protein were absent in control epithelium but were expressed significantly 48 h after inhalation.
  • EGF-R tyrosine kinase inhibitor BIBX1522
  • the head was decalcified with Surgipath (Decalcifier II, Surgical Medical Industries, Inc., Richmond, IL) for 4 - 5 days and rinsed in phosphate- buffered saline.
  • Surgipath Decalcifier II, Surgical Medical Industries, Inc., Richmond, IL
  • the nasal cavity was sectioned transversely at the level of the incisive papilla of the nasal palate.
  • the frontal tissue block was embedded in glycol methacrylate (JB 4 Plus, Polysciences, Inc., Warrington, PA), or in OCT compound (Sakura Finetek, U.S.A., Inc., Torrance, CA) for frozen sections.
  • Neutrophils were counted in high power fields of the epithelial layer stained with 3,3'-diaminobenzidine at magnification x400. The number of neutrophils within the nasal sepal epithelium (from the basement membrane to cell apices) was determined by counting the number of nuclear profiles per unit of basal lamina length.
  • the intracellular mucin in superficial epithelial secretory cells appears as oval-shaped, purple granules of varying sizes.
  • EGF-R and MUC5AC protein Immunolocalization of EGF-R and MUC5AC protein. Frozen sections from the paraformaldehyde-fixed nasal tissues were treated with 3 % H 2 0 2 /methanol to block endogenous peroxide and were incubated with a mouse monoclonal antibody to EGF-R (Calbiochem, San Diego, CA), or MUC5AC (NeoMarkers Inc., Fremont, CA) for 1 h at a dilution of 1 :100. Immunoreactive EGF- R or MUC5AC was visualized with the Vectastain Elite ABC kit (Vector Lab., Inc., Burhngame, CA) using 3,3-diaminobenzidine tetrahydrochlonde as a chromogen.
  • Vectastain Elite ABC kit Vector Lab., Inc., Burhngame, CA
  • Controls included the substitution of primary or secondary antibody with PBS.
  • Methods We studied pathogen-free rats, which normally have many goblet cells in the nasal septal epithelium. To determine the effect of aerosolized fMLP on goblet cell degranulation and on neutrophil migration into nasal mucosa epithelium, the animals were anesthetized with sodium pentobarbital (65 mg/kg, i.p.), and they received N-formyl-methionyl-leucyl-phenylalanine (fMLP; 10 ⁇ 5 M, Sigma, St. Louis, MO) in pyrogen-free saline intranasally by aerosol for 5 min.
  • fMLP N-formyl-methionyl-leucyl-phenylalanine
  • Aerosol exposure was accomplished by ventilating the animals with an ultrasonic nebulizer (PulmoSonic, DeVilbiss Co., Somerset, PA) that generated an aerosol mist at rate of 0.3 ml/min. Similarly, control animals were given saline aerosol alone intranasally.
  • an ultrasonic nebulizer PulmoSonic, DeVilbiss Co., Somerset, PA
  • EGF-R tyrosine kinase activation was evaluated on goblet cell re-granulation.
  • animals were pretreated intrapentoneally with an EGF-R tyrosine kinase inhibitor (BIBX1522, 15 mg/kg, generously provided by Boehringer Ingelheim Inc., Ingelheim, Germany) 30 min before the inhalation of fMLP and repeated twice a day.
  • an EGF-R tyrosine kinase inhibitor (BIBX1522, 15 mg/kg, generously provided by Boehringer Ingelheim Inc., Ingelheim, Germany) 30 min before the inhalation of fMLP and repeated twice a day.
  • non-stimulated rat nasal epithelium contains "stable", non- degranulating goblet cells containing mucin proteins.
  • Neutrophil chemoattractants e.g., fMLP
  • fMLP neutrophil chemoattractants
  • EGF-R EGF-receptors
  • EGF-R In pathogen-free rats goblet cells are "inactive" (ie, not degranulating) and EGF-R are down-regulated. When inflammation (eg, stimulation of neutrophil infiltration) causes GC degranulation and mucin secretion, up-regulation and activation of EGF-R re-supplies the airway epithelium with mucins.
  • inflammation eg, stimulation of neutrophil infiltration

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Abstract

L'invention concerne l'inhibition d'une hypersécrétion de mucus dans les poumons par l'administration d'un antagoniste du récepteur du facteur de croissance épidermique (EGF-R). L'antagoniste d'EGF-R peut se présenter sous la forme d'une petite molécule organique, d'un anticorps, ou d'une partie d'un anticorps qui se lie au récepteur d'EGF et le bloque. L'antagoniste d'EGF-R est de préférence administré par injection en quantité suffisante pour inhiber la formation de cellules caliciformes dans les voies respiratoires pulmonaires. De cette manière, on inhibe la dégranulation de cellules caliciformes qui entraîne la production de mucus dans les voies respiratoires. L'invention concerne également des tests de criblage d'agents candidats qui inhibent la prolifération de cellules caliciformes.
PCT/US1999/018696 1998-08-18 1999-08-17 Inhibition de la production de mucus dans les voies respiratoires par l'administration d'antagonistes d'egf-r WO2000010588A2 (fr)

Priority Applications (21)

Application Number Priority Date Filing Date Title
IL14085599A IL140855A0 (en) 1998-08-18 1999-08-17 Preventing airway mucus production by administration of egf-r antagonists
DE69939022T DE69939022D1 (de) 1998-08-18 1999-08-17 Epidermale wachstumsfaktor rezeptor antagonisten zur behandlung stark vermehrter schleimsekretion in der lunge
SK216-2001A SK287805B6 (sk) 1998-08-18 1999-08-17 Použitie antagonistu receptora epidermálneho rastového faktora (EGF-R) na výrobu lieku na liečenie nadmerného vylučovania hlienu v dýchacích cestách
PL351765A PL200940B1 (pl) 1998-08-18 1999-08-17 Zastosowanie antagonisty receptora naskórkowego czynnika wzrostowego (EGF-R) i postać farmaceutyczna do leczenia nadmiernego wydzielania śluzu dróg oddechowych
DK99943729T DK1119349T3 (da) 1998-08-18 1999-08-17 Epidermal vækstfaktor receptorantagonister til behandling af voldsomt foröget slimudskillelse i lungerne
SI9931016T SI1119349T1 (sl) 1998-08-18 1999-08-17 Antagonisti receptorja epidermalnega rastnega faktorja za zdravljenje hipersekrecije sluzi v pljučah
CA2337422A CA2337422C (fr) 1998-08-18 1999-08-17 Inhibition de la production de mucus dans les voies respiratoires par l'administration d'antagonistes d'egf-r
UA2001021134A UA73722C2 (en) 1998-08-18 1999-08-17 Treatment of mucus hypersecretion in lungs by administration of epidermal growth factor receptor (egf-r) antagonist and pharmaceutical formulation
BR9912672-9A BR9912672A (pt) 1998-08-18 1999-08-17 Prevenção de produção de muco no trato respiratório por administração de antagonistas de egf-r
HU0103894A HUP0103894A3 (en) 1998-08-18 1999-08-17 Preventing airway mucus production by administration of egf-r antagonists
JP2000565908A JP2002539072A (ja) 1998-08-18 1999-08-17 Ｅｇｆ−ｒアンタゴニストの投与による気道粘液産生の防止
EP99943729A EP1119349B1 (fr) 1998-08-18 1999-08-17 Inhibition de la production de mucus dans les voies respiratoires par l'administration d'antagonistes d'egf-r
AU56766/99A AU760766B2 (en) 1998-08-18 1999-08-17 Preventing airway mucus production by administration of EGF-R antagonists
EEP200100097A EE200100097A (et) 1998-08-18 1999-08-17 Hingamisteede lima tootmise tõkestamine EGF-R antagonistide manustamise teel
NZ509271A NZ509271A (en) 1998-08-18 1999-08-17 Epidermal growth factor receptor antagonists for treating hypersecretion of mucus in the lungs
EA200100136A EA004316B1 (ru) 1998-08-18 1999-08-17 Способ лечения гиперсекреции слизи
IL140855A IL140855A (en) 1998-08-18 2001-01-10 Use of an epidermal growth factor receptor (egf-r) antagonist for the preparation of pharmaceutical formulation for the treatment of airway mucus hypersecretion
BG105158A BG105158A (bg) 1998-08-18 2001-01-16 Предотвратяване образуването на слузести покрития(мускус) върху летателни писти с прилагане на противодействащо средство egf-r
NO20010749A NO327000B1 (no) 1998-08-18 2001-02-14 Anvendelse av epidermal vekstfaktorreseptor (EGF-R) antagonister
HR20010119A HRP20010119B1 (en) 1998-08-18 2001-02-16 Preventing airway mucus production by administration of egf-r antagonists
HK01106832A HK1036219A1 (en) 1998-08-18 2001-09-27 Epidermal growth factor receptor antagonists for treating hypersecretion of mucus in the lungs

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US6566324B2 (en) 1998-08-18 2003-05-20 The Regents Of The University Of California Preventing airway mucus production by administration of EGF-R antagonists
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US7354894B2 (en) 1998-08-18 2008-04-08 The Regents Of The University Of California Preventing airway mucus production by administration of EGF-R antagonists
US7358222B2 (en) 1998-08-18 2008-04-15 The Regents Of The University Of California Preventing airway mucus production by administration of EGF-R antagonists
US7531500B2 (en) 1998-08-18 2009-05-12 The Regents Of The University Of California Preventing airway mucus production by administration of EGF-R antagonists
US7700547B2 (en) 1998-08-18 2010-04-20 The Regents Of The University Of California Preventing airway mucus production by administration of EGF-R antagonists
US8048844B1 (en) 1998-08-18 2011-11-01 The Regents Of The University Of California Preventing airway mucus production by administration of EGF-R antagonists
US8071074B2 (en) 1998-08-18 2011-12-06 The Regents Of The University Of California Preventing airway mucus production by administration of EGF-R antagonists
WO2002005842A3 (fr) * 2000-07-14 2003-01-03 Univ California Administration d'antagonistes d'egf visant a empecher la production de mucus dans les voies respiratoires

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EE200100097A (et) 2002-06-17
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US6566324B2 (en) 2003-05-20
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IL140855A0 (en) 2002-02-10
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US6551989B2 (en) 2003-04-22
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US20080175797A1 (en) 2008-07-24
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US7358222B2 (en) 2008-04-15
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